Process for the fermentative preparation of D-pantothenic acid and/or its salts

ABSTRACT

A method for the fermentative preparation of D-pantothenic acid and/or its salts or feedstuffs additives containing these by the fermentation of microorganisms from the Enterobacteriaceae family in particular those which already produce D-pantothenic acid, wherein the nucleotide sequence(s) coding for the glyA gene is enhanced in the microorganisms, in particular is overexpressed.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a process for the fermentativepreparation of D-pantothenic acid and/or its salts or mixturescontaining these using microorganisms from the Enterobacteriaceaefamily, in which at least the glyA gene is enhanced.

[0003] 2. Description of the Background

[0004] Pantothenic acid is produced all over the world in amounts ofseveral thousand tons per year. It is used, inter alia, in humanmedicine, in the pharmaceutical industry and in the foodstuffs industry.A high proportion of the pantothenic acid produced is used for feedingeconomically useful animals such as poultry, and pigs. The demand forthis material is increasing.

[0005] Pantothenic acid can be prepared by chemical synthesis orbiotechnically by the fermentation of suitable microorganisms insuitable nutrient media. In the case of chemical synthesis,DL-pantolactone is an important precursor. This is prepared in amulti-step process from formaldehyde, isobutylaldehyde and cyanide, theracemic mixture is resolved in a subsequent process step, D-pantolactoneis condensed with 13-alanine and D-pantothenic acid is obtained in thisway.

[0006] The typical commercial form is the calcium salt of D-pantothenicacid. The calcium salt of the racemic mixture D,L-pantothenic acid isalso commonly available.

[0007] The advantage of fermentative preparation by microorganisms isthe direct formation of the desired stereoisomeric form, that is theD-form, which contains no L-pantothenic acid.

[0008] Various species of bacteria such as, e.g. Escherichia coli (E.coli), Arthrobacter ureafaciens, Corynebacterium erythrogenes,Brevibacterium ammoniagenes and also yeasts, such as e.g. Debaromycescastellii can, as shown in EP-A 0 493 060, produce D-pantothenic acid ina nutrient medium which contains glucose, DL-pantoic acid and β-alanine.Furthermore, EP-A 0 493 060 shows that, in the case of E. coli, theformation of D-pantothenic acid is improved by the amplification ofpantothenic acid biosynthesis genes from E. coli which are contained onthe plasmids pFV3 and pFV5, in a nutrient medium which contains glucose,DL-pantoic acid and β-alanine.

[0009] EP-A 0 590 857 and U.S. Pat. No. 5,518,906 describe mutantsderived from E. coli strain IF03547, such as FV5714, FV525, FV814,FV521, FV221, FV6051 and FV5069 which carry resistance to variousantimetabolites such as salicylic acid, a-ketobutyric acid,β-hydroxyaspartic acid, O-methylthreonine and a-ketoisovaleric acid.They produce pantoic acid in a nutrient medium which contains glucose,and D-pantothenic acid in a glucose and B-alanine-containing nutrientmedium. Furthermore, in EP-A 0 590 857 and U.S. Pat. No. 5,518,906, itis stated that the production of D-pantoic acid is improved in aglucose-containing nutrient media and the production of D-pantothenicacid is improved in a nutrient medium which contains glucose andβ-alanine after amplification, in the strains mentioned above, of thepantothenic acid biosynthesis genes panB, panC and panD, which should bepresent on the plasmid pFV31.

[0010] Furthermore, WO 97/10340 reports on the beneficial effect ofenhancing the ilvGM operon on the production of D-pantothenic acid.Finally, EP-A-1001027 reports on the effect of enhancing the panE geneon the formation of D-pantothenic acid. According to known procedures,D-pantothenic acid or the corresponding salt is isolated from thefermentation broth and purified (EP-A-0590857 and WO 96/33283) and thenused in purified form or the entire D-pantothenic acid-containing brothis dried EP-A-1050219) and used in particular as a foodstuffs additive.

[0011] In view of the increasing demand for D-panthothenic acid, thereremains a need for new methods of producing this material.

SUMMARY OF THE INVENTION

[0012] It is an object of the present invention to provide methods ofmaking D-pantothenic acid and salts thereof, as well as feedstuffsadditives containing the same.

[0013] The invention provides a process for the preparation ofD-pantothenic acid and/or its salts or foodstuffs additives whichcontain, in addition to these, further constituents from thefermentation by fermentation of microorganisms from theEnterobactericeae family, in particular those which already produceD-pantothenic acid, in which

[0014] (a) the nucleotide sequence(s) in the microorganisms coding forthe endogenous glyA gene is enhanced, in particular overexpressed, underconditions which are suitable for the production of serine hydroxymethyltransferase,

[0015] (b) D-pantothenic acid and/or its salts are enriched in themedium or in the cells of the microorganisms and

[0016] (c) the desired products are isolated after completion offermentation, wherein an amount of ≧0 to 100% of the biomass and/oroptionally further constituents of the fermentation broth are separated,

[0017] wherein the microorganisms produce D-pantothenic acid.

[0018] The invention also provides a process in which, after completionof fermentation, all or some of the biomass remains in the fermentationbroth and the broth obtained in this way is processed, optionally afterbeing concentrated, to give a solid mixture which contains D-pantothenicacid and/or its salts and which also contains other constituents of thefermentation broth.

[0019] Thus, the present invention provides a method of producingD-pantothenic acid and/or a salt thereof, comprising:

[0020] fermenting a microorganism of the family Enterobacteriaceae, inwhich the nucleotide sequence for the endogenous glyA gene is enhanced,in a medium suitable for the production of serine hydroxymethyltransferase, wherein the microorganism produces the D-pantothenic acidand/or a salt thereof.

[0021] The present invention also provides a method of producing afeedstuffs additive, comprising:

[0022] producing D-pantothenic acid and/or a salt thereof as describedabove, and

[0023] combining the D-pantothenic acid and/or a salt thereof with acarrier suitable for use in feedstuffs.

[0024] The present invention also provides a vector suitable forexpressing the glyA gene from E. coli which contains a promoter and thegene sequence.

[0025] The present invention also provides a microorganism from theEnterobacteriaceae family, transformed with the vector described above.

[0026] The present invention also provides a method for producingD-pantothenic acid and/or a salt thereof by fermenting the microorganismdescribed above.

[0027] In particular, yhe present invention also provides a method forproducing an animal feedstuffs additive, comprising:

[0028] (a) producing D-pantothenic acid or a salt thereof as describedabove, wherein the alkaline earth metal of the alkaline earth salt ismagnesium and/or calcium,

[0029] (b) optionally, removing water from the medium,

[0030] (c) separating the biomass formed during the fermentation in anamount of 0 to 100%,

[0031] (d) optionally, adding one or more magnesium and/or calcium saltsof D-pantothenic acid to the fermentation broths from (b), and

[0032] (e) producing the feedstuffs additive,

[0033] wherein the amount of the added one or more magnesium and/orcalcium salts of D-pantothenic acid is such that the amount thereof inthe feedstuffs additive is in the range from 1 about 20 to 80 wt. %based on the dry mass of the feedstuffs additive.

BRIEF DESCRIPTION OF THE FIGURES

[0034] A more complete appreciation of the invention and many of theattendant advantages thereof will be readily obtained as the samebecomes better understood by reference to the following detaileddescription when considered in connection with the accompanyingdrawings, wherein:

[0035]FIG. 1: Map of the plasmid pTrc99AglyA containing the glyA gene.

[0036]FIG. 2: Map of the plasmid pACYC184panBC containing the panBCgene.

[0037] Data relating to lengths are given as approximate values. Theabbreviations and names used are as follows:

[0038] Amp: Ampicillin resistance gene

[0039] Tc: Tetracyclin resistance gene

[0040] lacI: Gene for repressor protein of the trc promoter

[0041] Ptrc: trc promoter region, IPTG inducible

[0042] glyA: Coding region of the glyA gene

[0043] 5S: 5S rRNA region

[0044] rrnBT: rRNA terminator region

[0045] panB: Coding region of the panB gene

[0046] panC: Coding region of the panC gene

[0047] The abbreviations for the restriction enzymes are as follows:

[0048] BamHI: Restriction endonuclease from Bacillus amyloliquefaciens

[0049] BglII: Restriction endonuclease from Bacillus globigii

[0050] ClaI: Restriction endonuclease from Caryphanon latum

[0051] EcoRI: Restriction endonuclease from Escherichia coli

[0052] EcoRV: Restriction endonuclease from Escherichia coli

[0053] HindIII: Restriction endonuclease from Haemophilus influenzae

[0054] KpnI: Restriction endonuclease from Klebsiella pneumoniae

[0055] PstI: Restriction endonuclease from Providencia stuartii

[0056] PvuI: Restriction endonuclease from Proteus vulgaris

[0057] SacI: Restriction endonuclease from Streptomyces achromogenes

[0058] SalI: Restriction endonuclease from Streptomyces albus

[0059] SmaI: Restriction endonuclease from Serratia marcescens

[0060] XbaI: Restriction endonuclease from Xanthomonas badrii

[0061] XhoI: Restriction endonuclease from Xanthomonas holcicola.

DETAILED DESCRIPTION OF THE INVENTION

[0062] Whenever D-pantothenic acid, pantothenic acid or pantothenate arementioned in the following, these are intended to mean not only the freeacids but also the salts of D-pantothenic acid such as e.g. the calcium,sodium, ammonium or potassium salt.

[0063] In this connection, the expression “enhancement” describes theincrease in cellular activity of one or more enzymes or proteins in amicroorganism which are coded by the corresponding DNA by, for example,increasing the copy number of the gene or genes, using a strong promoteror a gene or allele which codes for a corresponding enzyme or proteinwith high activity and optionally combining these steps.

[0064] As a result of the enhancement step, in particularoverexpression, the activity or concentration of the correspondingprotein is generally increased by at least 10%, 25%, 50%, 75%, 100%,150%, 200%, 300%, 400% or 500%, at most up to 1000% or 2000%, withrespect to that of the wild type protein or the protein in the initialmicroorganism.

[0065] Microorganisms which are provided by the present invention canproduce D-pantothenic acid from glucose, saccharose, lactose, fructose,maltose, molasses, starch, cellulose or from glycerine and ethanol. Theyare members of the Enterobacteriaceae family, in particular from thegenus Escherichia. From the genus Escherichia, the species Escherichiacoli is mentioned in particular. Within the species Escherichia coli,the so-called K-12 strains such as e.g. the strain MG1655 or W3110(Neidhard et al.: Escherichia coli and Salmonella. Cellular andMolecular Biology (ASM Press, Washington D.C.)) or the Escherichia coliwild type strain IF03547 (Institut für Fermentation, Osaka, Japan) andmutants derived therefrom are suitable, these having the ability toproduce D-pantothenic acid.

[0066] Suitable D-pantothenic acid-producing strains from the genusEscherichia, in particular from the species Escherichia coli are forexample

[0067]Escherichia coli FV5069/pFV31

[0068]Escherichia coli FV5069/pFV202

[0069]Escherichia coli FE6/pFE80 and

[0070]Escherichia coli KE3.

[0071] It was found that Enterobacteriaceae, after overexpression of theglyA gene coding for serine hydroxymethyl transferase produceD-pantothenic acid in an improved way.

[0072] The nucleotide sequences in the glyA gene from Escherichia coliwere published by Plamann et al (Nucleic Acids Research11(7):2065-2075(1983)) and can also be obtained from the genome sequencefor Escherichia coli, under Accession Number AE000374, published byBlattner et al. (Science 277, 1453-1462 (1997).

[0073] The glyA gene described in the literature references cited abovecan be used in accordance with the invention. Furthermore, alleles ofthe glyA gene which are produced by degeneracy of the genetic code or byfunctionally neutral sense mutations may be used.

[0074] In order to produce an overexpression, the copy number of thecorresponding genes can be increased or the promoter and regulationregion or the ribosome bonding site which is located upstream of thestructure gene can be mutated. Expression cassettes which areincorporated upstream of the structure gene act in the same way. It isalso possible to increase expression during the course of fermentativeD-pantothenic acid production by means of inducible promoters.Expression is also improved by steps to extend the lifetime of m-RNA.Furthermore, by inhibiting degradation of the enzyme protein, enzymeactivity is also enhanced. The genes or gene structures may either bepresent in plasmids with different copy numbers or integrated andamplified in the chromosome. Alternatively, overexpression of the genesinvolved can be achieved by changing the composition of the medium andby culture management.

[0075] One skilled in the art will find instructions for this, interalia, in Chang and Cohen (Journal of Bacteriology 134:1141-1156 (1978)),in Hartley and Gregori (Gene 13:347-353 (1981)), in Amann and Brosius(Gene 40:183-190 (1985)), in de Broer et al. (Proceedings of theNational of Sciences of the United States of America 80:21-25 (1983)),in LaVallie et al. (BIO/TECHNOLOGY 11, 187-193 (1993)), inPCT/US97/13359, in Llosa et al. (Plasmid 26:222-224 (1991)), in Quandtand Klipp (Gene 80:161-169 (1989)), in Hamilton (Journal of Bacteriology171:4617-4622 (1989), in Jensen and Hammer (Biotechnology andBioengineering 58, 191-195 (1998) and in well-known textbooks ongenetics and molecular biology.

[0076] Plasmid vectors which are replicable in Enterobacteriaceae suchas e.g. cloning vectors derived from pACYC184 (Bartolome et al.; Gene102, 75-78 (1991)), pTrc99A (Amann et al.; Gene 69:301-315 (1988)) orpSC 101 derivatives (Vocke and Bastia, Proceedings of the NationalAcademy of Science USA 80 (21):6557-6561 (1983)) can be used. In oneprocess according to the invention, a strain transformed with a plasmidvector can be used, wherein the plasmid vector contains at least thenucleotide sequence coding for the glyA gene.

[0077] Furthermore, it may be advantageous for the production ofD-pantothenic acid using strains from the Enterobacteriaceae family, inaddition to enhancing the glyA gene, to enhance, in particular tooverexpress, separately or together one or more endogenous genesselected from the group

[0078] the ilvGM operon coding for acetohydroxy acid synthase II (WO97/10340)

[0079] the panB gene coding for ketopantoate-hydroxymethyl transferase(U.S. Pat. No. 5,518,906),

[0080] the pane gene coding for ketopantoate reductase (EP-A-1001027)

[0081] the panD gene coding for aspartate decarboxylase (U.S. Pat. No.5,518,906),

[0082] the panC gene coding for pantothenate synthetase (U.S. Pat. No.5,518,906),

[0083] the serC gene coding for phosphoserine transaminase (Duncan undCoggins, Biochemical Journal 234:49-57 (1986)) and

[0084] the gcvT, gcvH and gcvP genes coding for the glycine cleavagesystem (Okamura-Ikeda et al., European Journal of Biochemistry 216,539-548 (1993)).

[0085] Finally, it may be advantageous for the production ofD-pantothenic acid using strains of the Enterobacteriaceae family, inaddition to enhancing the glyA gene, to attenuate, in particular toswitch off or express at a lower level the following gene,

[0086] the avtA gene coding for transaminase C (EP-A-1001027).

[0087] The expression “attenuation” in this connection describes thereduction in, or switching off, of the intracellular activity of one ormore enzymes (proteins) in a microorganism which are coded by thecorresponding DNA by, for example, using a weak promoter or using a geneor allele which codes for a corresponding enzyme (protein) with a loweractivity or inactivates the corresponding gene or enzyme (protein) andoptionally combining these steps.

[0088] The activity or concentration of the corresponding protein isgenerally lowered to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5%of the activity or concentration of the wild type protein or of theactivity or concentration of the protein in the initial microorganism bythe attenuation step.

[0089] Furthermore, it may be advantageous for the production ofD-pantothenic acid, in addition to overexpressing the glyA gene, toswitch off undesired secondary reactions (Nakayama: “Breeding of AminoAcid Producing Microorganisms”, in: Overproduction of MicrobialProducts, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK,1982). In the process according to the invention, bacteria may be usedin which the metabolic pathways which reduce the formation ofD-pantothenic acid are at least partially switched off.

[0090] Microorganisms prepared according to the invention can becultivated in a batch process, in a fed batch process or in a repeatedfed batch process. Summaries of known cultivation methods are describedin the textbook by Chmiel (Bioprozesstechnik 1. Einführung in dieBioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in thetextbook by Storhas (Bioreaktoren und periphere Einrichtungen (ViewegVerlag, Braunschweig/Wiesbaden, 1994)).

[0091] The culture medium to be used has to satisfy the requirements ofthe particular strain in an appropriate manner. Descriptions of culturemedia for various microorganisms are given in the manual “Manual ofMethods for General Bacteriology” by the American Society forBacteriology (Washington D.C., USA, 1981). Sources of carbon which areused are sugar and carbohydrates such as e.g. glucose, saccharose,lactose, fructose, maltose, molasses, starch and cellulose, oils andfats such as e.g. soy oil, sunflower oil, groundnut oil and coconutbutter, fatty acids such as e.g. palmitic acid, stearic acid andlinoleic acid, alcohols such as e.g. glycerine and ethanol and organicacids such as e.g. acetic acid. These substances may be usedindividually or as a mixture.

[0092] Sources of nitrogen which are used may be organicnitrogen-containing compounds such as peptones, yeast extract, meatextract, malt extract, maize steep liquor, soy bean flour and urea orinorganic compounds such as ammonium sulfate, ammonium chloride,ammonium phosphate, ammonium carbonate and ammonium nitrate. The sourcesof nitrogen may be used individually or as a mixture.

[0093] Sources of phosphorus which may be used are phosphoric acid,potassium dihydrogen phosphate or dipotassium hydrogen phosphate or thecorresponding sodium-containing salts. Furthermore, the culture mediummust contain salts of metals, such as e.g. magnesium sulfate or ironsulfate, which are required for growth. Finally, essential growthsubstances such as amino acids and vitamins can be used in addition tothe substances mentioned above. Moreover, precursors of D-pantothenicacid such as aspartate, b-alanine, ketoisovalerate, ketopantoic acid orpantoic acid and optionally their salts can be added to the culturemedium. The feedstocks mentioned can be added to the culture in the formof a one-off batch or be fed to the culture medium in an appropriatemanner during cultivation.

[0094] Basic compounds such as sodium hydroxide, potassium hydroxide,ammonia or ammoniacal water or acid compounds such as phosphoric acid orsulfuric acid are used in a suitable way to control the pH of theculture.

[0095] It is also possible, to prepare the alkaline earth salts ofpantothenic acid, in particular the calcium salt, to add a suspension orsolution of an alkaline earth-containing inorganic compound, such as forexample calcium hydroxide, or an organic compound such as the alkalineearth salt of an organic acid, for example calcium acetate, continuouslyor batchwise during fermentation. The cation required to prepare thedesired alkaline earth salt of D-pantothenic acid is introduced directlyinto the fermentation broth in the desired amount in this way, generallyin the ratio of 0.8 to 1.2 to 1, with respect to the pantothenic acid,preferably in stoichiometric amounts.

[0096] Atifoaming agents such as e.g. polyglycol esters of fatty acidsare used to regulate the production of foam. To maintain the stabilityof plasmids, suitable selectively acting substances, e.g. antibiotics,may be added to the medium. In order to maintain the presence of aerobicconditions, oxygen or oxygen-containing gas mixtures such as e.g. airare introduced into the culture. The temperature of the culture isusually 25° C. to 45° C. and preferably 30° C. to 40° C. The culture iscontinued until a maximum of D-pantothenic has formed. This objective isusually achieved within 10 hours to 160 hours.

[0097] The D-pantothenic acid or the corresponding salts ofD-pantothenic acid contained in the fermentation broth may then beisolated and purified using known procedures. It is also possiblepreferably first to partly (³0 to 100%) or completely remove the biomassfrom the fermentation broth containing D-pantothenic acid and/or itssalts by known methods of separation such as, for example, centrifuging,filtering, decanting or a combination of these. However, it is alsopossible to leave all of the biomass in the fermentation broth. Ingeneral, the suspension or solution is preferably concentrated andworked up to produce a powder, for example using a spray dryer or afreeze drying unit. Then this powder is converted into a coarse-grained,very free-flowing, storable and largely dust-free product with aparticle size distribution of 20 to 2000 μm, in particular 100 to 140μm, using suitable compacting or granulating methods, e.g. alsopelletizing. The use of conventional organic or inorganic auxiliarysubstances, or supports such as starch, gelatin, cellulose derivativesor similar substances, such as are conventionally used in foodstuffs oranimal feed processing as -binders, gelling agents or thickeners, orother substances such as, for example, silicas, silicates or stearatesis advantageous when granulating or compacting.

[0098] Alternatively, the fermentation product, with or without otherconventional constituents from the fermentation broth, can be depositedonto an organic or inorganic support substance which is known andconventionally used in the foodstuffs processing sector such as, forexample, silicas, silicates, grist, bran, flour, starch, sugar or othersand/or stabilized with conventional thickeners or binders. Examples ofapplications and processes for this are described in the literature (DieMühle+Mischfuttertechnik 132 (1995) 49, page 817).

[0099] Optionally, in a suitable process step, D-pantothenic acid or thedesired salt of D-pantothenic acid or a preparation containing thesecompounds is added to the product in order to produce or adjust to thedesired concentration of pantothenic acid or the desired salt.

[0100] The desired concentration is generally in the range 20 to 80 wt.% (dry weight). This range includes all specific values and subrangestherebetween, such as 30, 40, 50, 60, and 70 wt. %.

[0101] The concentration of pantothenic acid can be determined usingknown chemical (Velisek; Chromatographic Science 60, 515-560 (1992)) ormicrobiological methods such as e.g. the Lactobacillus plantarum test(DIFCO MANUAL, 10th edition, p. 1100-1102; Michigan, USA).

[0102] A pure culture of the following microorganism was deposited atthe German Collection of Microorganisms and Cell Cultures (DSMZ,Braunschweig, Germany) on 8th September 2000 in accordance with theBudapest treaty:

[0103]Escherichia coli K12 strain FE6-1, as DSM 13721.

EXAMPLES

[0104] Having generally described this invention, a furtherunderstanding can be obtained by reference to certain specific exampleswhich are provided herein for purposes of illustration only and are notintended to be limiting unless otherwise specified.

[0105] The minimal (M9) and complete (LB) media for Escherichia coli aredescribed by J. H. Miller (A short course in bacterial genetics (1992),Cold Spring Harbor Laboratory Press). The isolation of plasmid DNA fromEscherichia coli and all the techniques for restriction, Klenow andalkaline phosphatase treatment are performed in accordance with Sambrooket al. (Molecular cloning—A laboratory manual (1989) Cold Spring HarborLaboratory Press). The transformation of Escherichia coli, if notdescribed differently, is performed in accordance with Chung et al.(Proceedings of the National Academy of Sciences of the United States ofAmerica USA (1989) 86: 2172-2175).

Example 1 Construction of the Expression Plasmid pTrc99AglyA

[0106] The glyA gene from E. coli K12 is amplified using the polymerasechain reaction (PCR) and synthetic oligonucleotides. Starting from thenucleotide sequence for the glyA gene in E. coli K12 MG1655 (AccessionNumber AE000341, Blattner et al. (Science 277, 1453-1462 (1997),) PCRprimers are synthesized (MWG Biotech, Ebersberg, Germany):

[0107] glyA1: 5′-GTTAGCTGAGTCAGGAGATG-3′

[0108] glyA2: 5′-TACGCTTATCAGGCCTACAC-3′

[0109] The chromosomal E. coli K12 MG1655 DNA used for the PCR isisolated according to data provided by the manufacturer using “QiagenGenomic-tips 100/G” (QIAGEN, Hilden, Germany). An approximately 1400 bpsize DNA fragment can be amplified with specific primers under standardPCR conditions (Innis et al. (1990) PCR Protocols. A Guide to Methodsand Applications, Academic Press) using Pfu DNA polymerase (PromegaCorporation, Madison, USA). The PCR product is ligated according to dataprovided by the manufacturer using the vector pCR-Blunt II-TOPO (ZeroBlunt TOPO PCR Cloning Kit, Invitrogen, Groningen, Netherlands) andtransformed in E. coli strain TOP10. The selection of cells carrying theplasmid is performed on LB agar which has been treated with 50 μg/mlkanamycin. After isolation of the plasmid DNA, the vector pCR-BluntII-TOPOglyA is cleaved with restriction enzymes HindIII and XbaI and theglyA fragment is isolated after separation in 0.8% agarose gel using theQIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). The vectorpTrc99A (Pharmacia Biotech, Uppsala, Sweden) is cleaved with the enzymesHindIII and XbaI and ligated with the isolated glyA fragment. E. colistrain XL1-Blue MRF′ (Stratagene, La Jolla, USA) is transformed with theligation mixture and cells carrying the plasmid are selected on LB agarwhich has been treated with 50 μg/ml ampicillin. Successful cloning canbe detected after plasmid DNA isolation by control cleavage with theenzyme SspI. The plasmid is called pTrc99AglyA (FIG. 1).

Example 2 Preparing the Strain FE6-1/pTrc99AglyA

[0110]E. coli strain FE6 is a valine-resistant mutant of E. coli K12MG1655 (U.S. Pat. No. 6,171,845) and is deposited as DSM12379 at theGerman Collection of Microorganisms and Cell Cultures (DSMZ,Braunschweig, Germany). Starting from FE6, spontaneous mutants areisolated after incubation at 37° C. on minimal agar which has beentreated with 2 g/l glucose and 1 g/l β-hydroxyaspartic acid. A selectedβ-hydroxyaspartic acid-resistant single colony is then incubated at 37°C. on minimal agar which contains 2 g/l glucose and 0.2 g/lO-methylthreonine. A mutant called FE6-1 is resistant to valine,a-ketoisovaleric acid, β-hydroxyaspartic acid and O-methylthreoninefollowing this step. The plasmid pTrc99AglyA is transformed in strainFE6-1 and cells carrying the plasmid are selected on LB agar which hasbeen treated with 50 μ/ml ampicillin. The strain obtained is calledFE6-1/pTrc99AglyA.

Example 3 Preparing the Strain FE6-1/pTrc99AglyA,pACYC 184panBC

[0111] The D-pantothenic acid-producing E. coli strain FV5069/pFV31 isdescribed in EP-A-0590857 and is deposited as FERM BP 4395 in accordancewith the Budapest treaty. The plasmid pFV31 is isolated fromFV5069/pFV31 and cleaved with restriction enzyme EcoRI. After separationin 0.8% agarose gel, the approximately 2600 bp size DNA fragment onwhich the panBC genes are present is isolated using the QIAquick GelExtraction Kit (QIAGEN, Hilden, Germany). The vector pACYC184 (Chang, A.C. Y. und Cohen, S. N., Journal of Bacteriology 134, 1141-1156 (1978);ATCC37033 (American Type Culture Collection, Manassas, USA)) is cleavedwith the enzyme EcoRI and ligated with the isolated panBC fragment. E.coli strain FE6-1 is transformed with the ligation mixture and cellswhich carry the plasmid are selected on LB agar which has been treatedwith 10 μg/ml tetracyclin. Successful cloning can be detected by controlcleavage with the enzymes EcoRV and EcoRI after plasmid DNA isolation.The plasmid is called pACYC184panBC (FIG. 2). The strainFE6-1/pTrc99AglyA described in example 2 is transformed with the plasmidpACYC184panBC. Selection is performed on LB agar which has been treatedwith 50 μg/ml ampicillin and 10 μg/ml tetracyclin. The strain producedin this way is called FE6-1/pTrc99AglyA, pACYC184panBC.

Example 4 Production of D-pantothenic Acid with Strains Derived fromFE6-1

[0112] Pantothenate production by E. coli strains FE6-1,FE6-1/pTrc99AglyA, FE6-1/pACYC184panBC, FE6-1/pTrc99AglyA, pACYC184panBCis checked in batch cultures of 10 ml which are contained in 100 mlconical flasks. For this, 10 ml of preculture medium with the followingcomposition: 2 g/l yeast extract, 10 g/l (NH₄)2SO₄, 1 g/l KH2PO4, 0.5g/l MgSO₄·₇H₂O, 15 g/l CaCO₃, 20 g/l glucose, is inoculated with asingle colony and incubated for 20 hours at 33° C. and 200 rpm in an ESRincubator from Kühner AG (Birsfelden, Switzerland). 200 μl portions ofthis preculture are each inoculated into 10 ml of production medium (25g/l (NH₄)₂SO₄, 2 g/l KH₂PO₄, 1 g/l MgSO₄·₇H₂O, 0.03 g/l FeSO₄·₇H₂O,0.018 g/l MnSO₄·₁H₂O, 30 g/l CaCO₃, 20 g/l glucose, 20 g/l β-alanine,250 mg/l thiamine) and incubated for 48 hours at 37° C. During theincubation of FE6-1/pTrc99AglyA, 50 mg/l ampicillin are also added tothe media, during the incubation of FE6-1/pACYC184panBC, 10 mg/ltetracyclin are also added to the media and during the incubation ofFE6-1/pTrc99AglyA,pACYC184panBC, 50 mg/l ampicillin and 10 mg/ltetracyclin are also added to the media. After incubation, the opticaldensity (OD) of the culture suspension is determined at a testwavelength of 660 nm using a LP2W photometer from the Dr. Lange Co.(Düsseldorf, Germany).

[0113] Then the concentration of D-pantothenate formed in the sterilefiltered culture supernatant liquid is determined using theLactobacillus plantarum ATCC8014 pantothenate assays in accordance withdata from DIFCO (DIFCO MANUAL, 10th Edition, p. 1100-1102; Michigan,USA). The calcium salt of D(+)-pantothenic acid hydrate (cataloguenumber 25,972-1, Sigma-Aldrich, Deisenhofen, Germany) is used forcalibration purposes.

[0114] Table 1 gives the results of the test. TABLE 1 OD PantothenateStrain (660 nm) mg/l FE6-1 10.6 19 FE6-1/pTrc99AglyA 9.2 39 FE6- 8.4 5401/pACYC184panBC FE6-1/pTrc99AglyA, 9.4 820 pACYC184panBC

[0115] The publications cited above are incorporated herein byreference.

[0116] Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

[0117] This application is based on German Patent Application Serial No.101 06 461.6, filed on Feb. 13, 2001, which is incorporated herein byreference.

1. A method of producing D-pantothenic acid and/or a salt thereof,comprising: fermenting a microorganism of the family Enterobacteriaceae,in which the nucleotide sequence for the endogenous glyA gene isenhanced, in a medium suitable for the production of serinehydroxymethyl transferase, wherein the microorganism produces theD-pantothenic acid and/or a salt thereof.
 2. The method of claim 1,wherein the medium is enriched in the D-pantothenic acid and/or a saltthereof and/or the cells of the microorganism are enriched in theD-pantothenic acid and/or a salt thereof.
 3. The method of claim 1,wherein the nucleotide sequence for the endogenous glyA gene is enhancedis overexpressed.
 4. The method of claim 1, further comprising isolatingat least a portion of the D-pantothenic acid and/or a salt thereof fromthe medium.
 5. The method of claim 1, further comprising isolating atleast a portion of the biomass from the medium.
 6. The method of claim1, wherein the fermentation is performed in the presence of at least onealkaline earth salt, which is supplied continuously or batchwise to themedium, and a product containing an alkaline earth salt of D-pantothenicacid is produced.
 7. The method of claim 1, wherein the microorganismbelongs to the genus Escherichia.
 8. The method of claim 1, wherein themicroorganism is an Escherichia coli.
 9. The method of claim 1, whereinone or more of the endogenous genes selected from the group consistingof the ilvGM operon coding for acetohydroxy acid synthase II, the panBgene coding for ketopantoate hydroxymethyl transferase, the panE genecoding for ketopantoate reductase, the panD gene coding for aspartatedecarboxylase, the panC gene coding for pantothenate synthetase, theserC-Gene coding for phosphoserine transaminase, and the gcvT, gcvH andgcvP genes coding for the glycine cleavage system, are enhanced in themicroorganism.
 10. The method of claim 1, wherein the metabolic pathwayswhich reduce the formation of D-pantothenic acid are at least partiallyswitched off in the microorganism.
 11. The method of claim 10, whereinthe avtA gene coding for transaminase C is attenuated.
 12. The method ofclaim 11, wherein the avtA gene is switched off or expressed at a lowlevel.
 13. The method of claim 1, wherein the activity or concentrationof the glyA gene product is increased by 10 to 2000%, with respect tothat of the wild type protein or the protein in the initialmicroorganism.
 14. The method of claim 1, wherein transformed strainsare used in which the glyA gene is present in plasmids or is integratedin the chromosome, and is enhanced.
 15. A method of producing afeedstuffs additive, comprising: producing D-pantothenic acid and/or asalt thereof as recited in claim 1, and combining the D-pantothenic acidand/or a salt thereof with a carrier suitable for use in feedstuffs. 16.The method of claim 15, wherein the carrier is selected from the groupconsisting of silicic acids, silicates, grist, bran, flour, starches,and sugars.
 17. The method of claim 15, wherein the D-pantothenic acidand/or a salt thereof also contains at least a portion of the biomassfrom the fermentation used to produce the D-pantothenic acid and/or asalt thereof.
 18. A vector suitable for expressing the glyA gene from E.coli which contains a promoter and the gene sequence.
 19. The vector ofclaim 18, which is the plasmid pTrc99AglyA shown in FIG.
 1. 20. Amicroorganism from the Enterobacteriaceae family, transformed with thevector of claim
 18. 21. A microorganism from the Enterobacteriaceaefamily, transformed with the vector in accordance with claim
 19. 22. Amethod for producing D-pantothenic acid and/or a salt thereof byfermenting the microorganism of claim
 20. 23. A method for producingD-pantothenic acid and/or a salt thereof by fermenting the microorganismof claim
 20. 24. The method of claim 1, further comprising:separating >0 to 100% of the biomass and/or optionally the contents ofthe medium from the medium, optionally, concentrating the resultingmixture, and converting the D- pantothenic acid and/or a salt thereofinto a free-flowing form.
 25. The method of claim 24, further comprisingproducing a free-flowing animal feedstuffs additive with a particle sizedistribution of 20 to 2000 μm from the free-flowing D-pantothenic acidand/or a salt thereof.
 26. The method of claim 25, wherein thefeedstuffs additive has a particle size distribution of 100 to 1400 μm.27. A method for producing an animal feedstuffs additive, comprising:(a) producing D-pantothenic acid or a salt thereof as recited in claim6, wherein the alkaline earth metal of the alkaline earth salt ismagnesium and/or calcium, (b) optionally, removing water from themedium, (c) separating the biomass formed during the fermentation in anamount of 0 to 100%, (d) optionally, adding one or more magnesium and/orcalcium salts of D-pantothenic acid to the fermentation broths from (b),and (e) producing the feedstuffs additive, wherein the amount of theadded one or more magnesium and/or calcium salts of D-pantothenic acidis such that the amount thereof in the feedstuffs additive is in therange from about 20 to 80 wt. % based on the dry mass of the feedstuffsadditive.
 28. The method of claim 27, wherein the animal feedstuffsadditive is in powder form.
 29. The method of claim 27, wherein theanimal feedstuffs additive is in granular form.
 30. The method of claim24, wherein an animal feedstuffs additive is obtained in the desiredpowder or granular form from the fermentation medium, optionally afterthe addition of D-pantothenic acid and/or a salt thereof and,optionally, after the addition of organic or inorganic auxiliarysubstances, by drying and compacting, or spray drying, or spray dryingand granulating, or spray drying and pelletizing.